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- PDB-4lnu: Nucleotide-free kinesin motor domain in complex with tubulin and ... -

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Basic information

Entry
Database: PDB / ID: 4lnu
TitleNucleotide-free kinesin motor domain in complex with tubulin and a DARPin
Components
  • Designed ankyrin repeat protein (DARPIN) D1
  • Kinesin-1 heavy chainKinesin
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsCELL CYCLE/MOTOR PROTEIN / ALPHA-TUBULIN / APO-KINESIN / BETA-TUBULIN / DARPIN / KINESIN / MICROTUBULE / TUBULIN / CELL CYCLE-MOTOR PROTEIN complex
Function / homology
Function and homology information


cytoplasm organization / cytolytic granule membrane / plus-end-directed vesicle transport along microtubule / anterograde neuronal dense core vesicle transport / anterograde dendritic transport of neurotransmitter receptor complex / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / vesicle transport along microtubule / lysosome localization ...cytoplasm organization / cytolytic granule membrane / plus-end-directed vesicle transport along microtubule / anterograde neuronal dense core vesicle transport / anterograde dendritic transport of neurotransmitter receptor complex / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / vesicle transport along microtubule / lysosome localization / positive regulation of potassium ion transport / natural killer cell mediated cytotoxicity / Kinesins / plus-end-directed microtubule motor activity / RHO GTPases activate KTN1 / stress granule disassembly / COPI-dependent Golgi-to-ER retrograde traffic / mitochondrion transport along microtubule / ciliary rootlet / centrosome localization / kinesin complex / synaptic vesicle transport / microtubule motor activity / microtubule-based movement / Insulin processing / centriolar satellite / Signaling by ALK fusions and activated point mutants / cytoplasmic microtubule / Nuclear events stimulated by ALK signaling in cancer / microtubule-based process / phagocytic vesicle / axon cytoplasm / MHC class II antigen presentation / dendrite cytoplasm / regulation of membrane potential / cellular response to interleukin-4 / axon guidance / positive regulation of synaptic transmission, GABAergic / positive regulation of protein localization to plasma membrane / structural constituent of cytoskeleton / microtubule cytoskeleton organization / cellular response to type II interferon / double-stranded RNA binding / microtubule binding / vesicle / microtubule / cadherin binding / GTPase activity / ubiquitin protein ligase binding / protein-containing complex binding / GTP binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Kinesin motor domain / Kinesin / Helix hairpin bin / Kinesin-like protein / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / Ankyrin repeat-containing domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain ...Kinesin motor domain / Kinesin / Helix hairpin bin / Kinesin-like protein / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / Ankyrin repeat-containing domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / 60s Ribosomal Protein L30; Chain: A; / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Helix Hairpins / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Kinesin-1 heavy chain / Tubulin alpha chain
Similarity search - Component
Biological speciesArtificial gene (others)
Homo sapiens (human)
Ovis aries (sheep)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.19 Å
AuthorsCao, L. / Gigant, B. / Knossow, M.
CitationJournal: Nat Commun / Year: 2014
Title: The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement
Authors: Cao, L. / Wang, W. / Jiang, Q. / Wang, C. / Knossow, M. / Gigant, B.
History
DepositionJul 12, 2013Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Dec 3, 2014Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tubulin alpha chain
B: Tubulin beta chain
D: Designed ankyrin repeat protein (DARPIN) D1
K: Kinesin-1 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,23323
Polymers154,6224
Non-polymers2,61119
Water11,403633
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13040 Å2
ΔGint-197 kcal/mol
Surface area49070 Å2
MethodPISA
Unit cell
Length a, b, c (Å)73.400, 83.010, 83.307
Angle α, β, γ (deg.)116.16, 105.08, 97.57
Int Tables number1
Space group name H-MP1

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Components

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Protein , 4 types, 4 molecules ABDK

#1: Protein Tubulin alpha chain


Mass: 50204.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / Organ: BRAIN / References: UniProt: W5QC38*PLUS
#2: Protein Tubulin beta chain


Mass: 50071.926 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / Organ: BRAIN / References: UniProt: D0VWY9*PLUS
#3: Protein Designed ankyrin repeat protein (DARPIN) D1


Mass: 18068.439 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Artificial gene (others) / Plasmid: PDST067 (PQE30 DERIVATIVE) / Production host: Escherichia coli (E. coli) / Strain (production host): XL1BLUE
#4: Protein Kinesin-1 heavy chain / Kinesin / Conventional kinesin heavy chain / Ubiquitous kinesin heavy chain / UKHC


Mass: 36276.746 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 1-325 / Mutation: C7S, C65A, C168A, C174S, C294A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIF5B / Plasmid: PET28 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P33176

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Non-polymers , 7 types, 652 molecules

#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: SO4
#8: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#9: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID / MES (buffer)


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#10: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#11: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 633 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsTHE SEQUENCE OF KINESIN (CHAIN K) INCLUDED THE FOLLOWING MUTATIONS: C7S, C65A, C168A, C174S AND ...THE SEQUENCE OF KINESIN (CHAIN K) INCLUDED THE FOLLOWING MUTATIONS: C7S, C65A, C168A, C174S AND C294A. THE CONSTRUCT IS KNOWN AS CYS-LITE (RICE ET AL, NATURE 1999, VOL 402: 778-84). IT WAS TRUNCATED AFTER RESIDUE 325 FOR THIS STUDY. FOR TUBULIN THE BOVINE BRAIN TUBULIN SEQUENCE WAS USED FOR REFINEMENT BECAUSE THE SEQUENCE OF OVINE BRAIN TUBULIN IS NOT AVAILABLE. FOR ALPHA-TUBULIN (CHAIN A) THE ALPHA 1B ISOTYPE SEQUENCE (NCBI NP_001108328.1) WAS USED. FOR BETA-TUBULIN (CHAIN B), THERE ARE THREE MAJOR ISOTYPES EXPRESSED IN THE BRAIN (BANERJEE AT AL, JBC 1988, VOL 263:3029-34). THE BETA 2B ISOTYPE SEQUENCE (NCBI NP_001003900.1) WAS USED BUT POINT MUTATIONS WERE INTRODUCED WHEN POSSIBLE TO TAKE INTO ACCOUNT THE ISOTYPE DIVERSITY.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.74 Å3/Da / Density % sol: 55 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 6.5
Details: PEG, MES BUFFER, 0.2M AMMONIUM SULFATE, pH 6.50, VAPOR DIFFUSION, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.979 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 17, 2013
RadiationMonochromator: CHANNEL CUT SI111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.19→43.45 Å / Num. obs: 77650 / % possible obs: 92 % / Redundancy: 3.6 % / Biso Wilson estimate: 42.69 Å2 / Rsym value: 0.074 / Net I/σ(I): 12.5
Reflection shellResolution: 2.19→2.31 Å / Redundancy: 2.5 % / Mean I/σ(I) obs: 2.3 / Rsym value: 0.491 / % possible all: 58.4

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Processing

Software
NameVersionClassification
PHASERphasing
BUSTER2.10.0refinement
XDSdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1BG2, 4DRX

1bg2

4drx


Resolution: 2.19→43.45 Å / Cor.coef. Fo:Fc: 0.9542 / Cor.coef. Fo:Fc free: 0.9382 / Occupancy max: 1 / Occupancy min: 0.1 / SU R Cruickshank DPI: 0.204 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.201 / SU Rfree Blow DPI: 0.163 / SU Rfree Cruickshank DPI: 0.166
RfactorNum. reflection% reflectionSelection details
Rfree0.1948 3881 5 %RANDOM
Rwork0.1554 ---
obs0.1574 77603 92.27 %-
Displacement parametersBiso max: 208.07 Å2 / Biso mean: 59.4878 Å2 / Biso min: 21.25 Å2
Baniso -1Baniso -2Baniso -3
1-6.4824 Å2-4.974 Å2-2.0909 Å2
2--2.0735 Å2-6.2278 Å2
3----8.5559 Å2
Refine analyzeLuzzati coordinate error obs: 0.275 Å
Refinement stepCycle: LAST / Resolution: 2.19→43.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10378 0 152 633 11163
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d3750SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes292HARMONIC2
X-RAY DIFFRACTIONt_gen_planes1575HARMONIC5
X-RAY DIFFRACTIONt_it10773HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion1412SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact12980SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d10773HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg14605HARMONIC21.17
X-RAY DIFFRACTIONt_omega_torsion3.05
X-RAY DIFFRACTIONt_other_torsion19.56
LS refinement shellResolution: 2.19→2.25 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.2538 156 4.98 %
Rwork0.206 2979 -
all0.2082 3135 -
obs--92.27 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.0258-0.61480.17041.72810.01550.36880.03380.0219-0.3871-0.0558-0.02730.02090.071-0.0364-0.0065-0.1234-0.00610.0447-0.16690.0237-0.005926.7016-20.326814.5155
22.1314-0.4082-0.15281.31930.38380.867-0.0667-0.35630.04230.19830.09510.00370.07240.1312-0.0284-0.1482-0.01070.0182-0.09470.0132-0.1079-0.71288.108328.9183
34.0724-0.7096-0.00762.9785-0.07390.8656-0.0582-0.42020.8850.0822-0.02080.0012-0.1053-0.07030.079-0.1610.0029-0.005-0.1718-0.09020.148-33.206624.26123.0524
44.3862-1.22810.83683.1377-0.90463.19510.58641.78870.0842-0.4502-0.59930.0290.07140.25210.0129-0.25390.2073-0.00320.29860.0907-0.50672.423312.9567-13.5866
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A1 - 502
2X-RAY DIFFRACTION2{ B|* }B1 - 502
3X-RAY DIFFRACTION3{ D|* }D13 - 169
4X-RAY DIFFRACTION4{ K|* }K8 - 323

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